All viruses depend on the host's translation (protein synthesis) machinery. For this reason, host cells have evolved numerous antiviral mechanisms that shut down or otherwise regulate translation. In the molecular arms race, viruses, in turn, have evolved ways to bypass host translational control. It is this essential step in virus replication that is the focus of this proposal. Host mRNAs contain a 5' cap structure and a 3' poly(A) tail that interact with translation initiation factors which recruit the ribosome to the mRNA. In contrast, the RNAs of most RNA viruses are uncapped, so they have evolved RNA structures that recruit the ribosome by noncanonical, cap-independent mechanisms. Many uncapped plant viral RNAs contain a cap-independent translation element (CITE) in the 3' untranslated region that facilitates efficient ribosome entry at the 5' end of the genome. In NIH-funded research the PI's lab showed that this is facilitated by long-distance base pairing between the 3' CITE, which binds a translation initiation factor, and the 5' untranslated region. Unanswered is how the CITE RNA structure causes it to bind a translation initiation factor with high affinity, leading to recruitment of the ribosome. Here, a variety of approaches will be applied to determine the structural requirements of two unrelated 3' CITEs, and the translation factors with which they interact. These include (i) the Barley yellow dwarf virus-like translation element (BTE) which binds and requires initiation factor eIF4G and not eIF4E; and (ii) the Panicum mosaic virus-like translation element (PTE), which binds and requires eIF4E - a protein known previously to bind only to the 5' cap structure. The three aims all can be performed independently, but the knowledge gained from each will feed into the other two aims. Aim I uses multiple, factor-depletable translation systems of mutant CITEs and mutant cognate translation factors with which they interact. This will reveal the key nucleotides and amino acids required for interaction and translation function. The second aim uses a variety of methods to measure the interactions of the mutant CITEs with mutant translation factors. The third aim will determine CITE structure at high resolution by ion-dependent RNA folding and X-ray crystallography methods. This project will provide a new understanding of the way in which viruses take over the cell, which may, in turn, suggest potential targets for antiviral drugs. Although this work focuses on model plant viruses, many growing human viruses such as dengue and hepatitis C viruses use similar mechanisms. Also, this work will shed new light on how the translational machinery works, and the translation system is extremely highly conserved between plants and animals. For example, the PTE functions in mammalian cells and we will use human cells and extracts to study how it uses eIF4E to usurp the ribosomes. Over-active eIF4E causes tumors and restriction of its function inhibits many types of cancers. The tightly binding PTE RNA may provide structural knowledge for design of eIF4E-inhibiting cancer therapeutics.